This is the first revision of a phase I STTR proposal to develop new therapies for the treatment of rheumatoid arthritis (RA). It is collaboration between a biotechnology company, Adenosine Therapeutics, LLC (ATL) based in Charlottesville, VA, and the University of Virginia laboratory of Dr. Donald L. Kimpel, a Rheumatologist experienced in the study of RA. Human RA is the most common form of inflammatory arthritis and is a chronic disorder of unknown origin with a variable course of disease. The majority of patients with RA have a progressive course which leads to destruction of joint tissue, instability of joints, loss of function and mobility, and increased mortality. Adenosine Therapeutics, LLC has synthesized, patented, and characterized a series of novel, orally available, and selective A2A adenosine receptor (A2AR) agonists that are effective in reducing inflammation in a variety of animal models. Our preliminary data suggest that 1 of these compounds, ATL313, is effective in reducing inflammation and joint damage in a well established rat model of RA. Current drugs used in the management of RA have a variety of side-effects, including cardiovascular complications. Our preliminary dosing and toxicity studies demonstrate no significant side-effects at doses which suppress inflammatory arthritis. Thus, we will further evaluate the effectiveness of the A2AR as a target for RA treatment. The first Aim is to synthesize adequate quantities of ATL313 and its deuterated standard to support these preliminary efficacy and metabolic studies. The second Aim is to characterize the products of the metabolism of these compounds by mouse, rat, dog, and human microsomes and hepatocytes and to confirm the presence of these metabolites in vivo in mice using LC/MS (HPLC coupled Mass Spectroscopy). Our third Aim is to evaluate the efficacy of ATL313 in a collagen-induced model of RA in rats (3A) and mice (3B). We have generated preliminary data that demonstrates the efficacy of ATL313 in a streptococcal cell wall model of arthritis and propose to validate this by comparison to the collagen model. The fourth Aim is to confirm that the A2AR mediates ATL313 activity in the collagen model by co-administering the selective A2AR antagonist ZM241385 (ZM) and to determine the duration of protection afforded by ATL313 by observing animals 60 days after cessation of drug administration. [unreadable] [unreadable] [unreadable]